The viral Gag proteins control many aspects of the HIV-1 replication cycle. The Gag precursor protein drives the assembly of virus particles in the infected cell, and, through putative interactions with the transmembrane envelope (Env) glycoprotein gp41, directs Env incorporation into virus particles. Following infection, the Gag proteins play a central role in uncoating and assist in the reverse transcription process. To characterize functions of the HIV-1 Gag matrix (MA) protein, we have introduced over 80 single and double amino acid mutations throughout MA. Biological and biochemical analysis identified classes of mutations that displayed defects at various stages of the virus life cycle. We have also introduced a number of premature termination codons throughout the Gag precursor protein to generate a series of truncated Gag proteins. Analysis of the ability of these truncated Gags to bind membrane, direct assembly and release of virus-like particles, and to interact with full-length Gag has provided information concerning the domains in Gag responsible for membrane binding and virus assembly. We observed a critical role for residues near the N-terminus of MA in binding of Gag to membrane, and observed that truncated Gag proteins containing MA and the C-terminal domain of capsid (CA) bound membrane as efficiently as full-length Gag.Mutations at MA amino acid 20 caused a defect early in the virus life. Viral DNA synthesis was also diminished in endogenous RT assays, and Gag proteins containing MA residue 20 substitutions exhibited increased membrane binding capacity relative to wild-type. A viral revertant of a residue 20 mutant maintained the position 20 change and acquired second-site changes at MA residues 73 and 82. Activity of the revertant virus in the endogenous RT assay was restored to nearly wild-type levels. Intriguingly, when virions were pseudotyped with the murine leukemia virus Env glycoproteins, the revertant changes in MA blocked the ability of HIV-1 protease to cleave the cytoplasmic domain of the murine leukemia virus transmembrane Env protein, thereby abolishing virus infectivity.Lentiviruses, including HIV-1, possess transmembrane Env glycoproteins with unusually long cytoplasmic tails. We have observed a strong cell-type-dependent requirement for the long cytoplasmic tail in virus replication; some cell lines are permissive and others non-permissive for large gp41 cytoplasmic tail deletions. This requirement is not imposed at the level of virus entry, as mutants lacking the gp41 cytoplasmic tail efficiently infect both permissive and non-permissive cell types. Using vesicular stomatitis virus G protein pseudotypes, we developed a high transient expressions system that enabled us to biochemically assess Env incorporation in non- permissive cell types. This analysis demonstrated that the block in non-permissive cells was imposed at the level of Env incorporation. These results indicate that the long gp41 cytoplasmic tail plays a critical role in Env incorporation in a cell-type dependent manner, implying a role for host factors in the Env incorporation process. A prominent structural feature of the HIV-1 gp41 cytoplasmic domain is the presence of two predicted alpha-helices in the central (helix 2) and C-terminal (helix 1) portions of the cytoplasmic domain. To characterize these domains, we have introduced a number of truncations, deletions, and single and double amino acid substitution mutations throughout both alpha-helical domains. The effects on Env expression, Env incorporation into virions, virus infectivity in single-cycle assays, virus replication in T-cells, and syncytium formation were assessed. The results indicate that the gp41 cytoplasmic tail helical domains play an important role in Env incorporation and virus infectivity. Viral revertants of several alpha-helix 2 mutants were isolated and characterized. Interestingly, one of the revertant changes mapped to MA; the second-site change in MA compensated for the Env incorporation defect imposed by the gp41 mutation. These results support the existence of a MA/gp41 interaction during Env incorporation. - Gag, matrix, envelope glycoproteins, HIV-1, retrovirus, virus assembly, membrane binding, gp41, Gag targeting.